Biochem. J. (2012) 446, 9–21 (Printed in Great Britain)
9
doi:10.1042/BJ20111144
A functional interaction between the MAGUK protein hDlg and the gap
junction protein connexin 43 in cervical tumour cells
Alasdair I. MACDONALD*, Peng SUN*1 , Hegel HERNANDEZ-LOPEZ*, Trond AASEN*2 , Malcolm B. HODGINS†,
Michael EDWARD†, Sally ROBERTS§, Paola MASSIMI‡, Miranda THOMAS‡, Lawrence BANKS‡ and Sheila V. GRAHAM*3
*MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow,
Glasgow G12 8TT, Scotland, U.K., †Section of Dermatology, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G11 6NU, Scotland,
U.K., ‡International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy, and §CRUK Birmingham Cancer Centre, School of Cancer Sciences,
University of Birmingham, Birmingham B15 2TT, U.K.
Gap junctions, composed of Cxs (connexins), allow direct
intercellular communication. Gap junctions are often lost during
the development of malignancy, although the processes behind
this are not fully understood. Cx43 is a widely expressed Cx
with a long cytoplasmic C-terminal tail that contains several
potential protein-interaction domains. Previously, in a model
of cervical carcinogenesis, we showed that the loss of gap
junctional communication correlated with relocalization of
Cx43 to the cytoplasm late in tumorigenesis. In the present
study, we demonstrate a similar pattern of altered expression for
the hDlg (human discs large) MAGUK (membrane-associated
guanylate kinase) family tumour suppressor protein in cervical
tumour cells, with partial co-localization of Cx43 and hDlg in
an endosomal/lysosomal compartment. Relocalization of these
proteins is not due to a general disruption of cell membrane
integrity or Cx targeting. Cx43 (via its C-terminus) and hDlg
interact directly in vitro and can form a complex in cells. This
novel interaction requires the N- and C-termini of hDlg. hDlg is
not required for Cx43 internalization in W12GPXY cells. Instead,
hDlg appears to have a role in maintaining a cytoplasmic pool of
Cx43. These results demonstrate that hDlg is a physiologically
relevant regulator of Cx43 in transformed epithelial cells.
INTRODUCTION
cycle of synthesis, intracellular trafficking and degradation [4].
A body of evidence has accumulated to show that GJIC may
be lost during malignant progression, for instance, in HPV
(human papillomavirus)-positive cervical cancer [9–12]. Cx43,
the most widespread Cx and a major component of gap junctions
in stratified epithelia, has been seen to be down-regulated in
epithelial carcinoma cells [13,14]. Nevertheless, the steps leading
to the loss of GJIC in cancer remain largely unknown.
At cell-contact sites, the MAGUK [membrane-associated GUK
(guanylate kinase)] family, members of which have several
protein-interaction domains, can form protein scaffolds and
comprise macromolecular complexes with their protein partners,
which are thought to be involved in cell-signalling cascades
and cell-morphology organization [15,16]. The terminal five
amino acids of Cx43 have been shown to bind to the middle of
three adjacent PDZ domains in ZO-1 (zona occludens-1) protein
[17,18]. ZO-1 was first found to regulate gap junction size [19]
and more recently to control the rate of formation of gap junctions
from connexons in the plasma membrane [20]. Cx45 also interacts
with ZO-1 through its C-terminal domain [21,22], but the role of
this interaction has not been fully elucidated.
In contrast, Cx32 does not interact with ZO-1 [23], but
instead interacts with the C-terminal SH3 (Src homology 3)
domain of another MAGUK protein, the human homologue of
the Drosophila discs large protein (hDlg) [24]. In Drosophila,
Intercellular junctions and various adhesion molecules in
mammals regulate cell–cell interactions. The membrane junction
complexes comprise gap junctions, tight junctions and adherens
junctions [1]. Gap junctions are specialized cell membrane
channels that allow direct intercellular diffusion of critical
regulatory ions and small molecules <1 kDa between the cytoplasmic interiors of contiguous cells, providing a direct
co-ordination of electrical and metabolic functions, proliferation
and differentiation [2,3]. Gap junction channels are formed
of paired connexons, each composed of six Cx (connexin)
subunits, docking with their counterparts in the membrane of the
neighbouring cell to form a continuous aqueous channel [4]. The
core of each Cx is buried in the plasma membrane and consists of
four hydrophobic α-helices connected by short loops. However,
Cx43 (and other members of the α connexon subgroup [5]) have
a long cytoplasmic C-terminal tail that appears to be involved in
several protein–protein interactions [4].
GJIC (gap junctional intercellular communication) is tightly
regulated by many mechanisms including Cx transcriptional and
post-transcriptional control [6], and also post-translational
regulation related to Cx protein turnover, trafficking and
hemichannel and gap junction activity [3,7,8]. Cxs have a short
half-life estimated to be 1–5 h and are involved in a constant
Key words: cervical cancer, connexin 43 (Cx43), gap junction,
human Dlg (hDlg), human papillomavirus (HPV).
Abbreviations used: Cx, connexin; DAPI, 4 , 6-diamidino-2-phenylindole; Dlg, discs large; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic
reticulum; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GJIC, gap junctional intercellular communication; GST,
glutathione transferase; GUK, guanylate kinase; hDlg, human Dlg; HPV, human papillomavirus; HRP, horseradish peroxidise; IP, immunoprecipitation; IPTG,
isopropyl β-D-thiogalactopyranoside; KGM, keratinocyte growth medium; MAGUK, membrane-associated guanylate kinase; MPR, mannose 6-phosphate
receptor; NP-40, Nonidet P40; PBST, PBS containing 0.1 % Tween 20; SH3, Src homology 3; siRNA, small interfering RNA; TopBP1, topoisomerase binding
protein 1; ZO-1, zona occludens-1.
1
Present address: MD Anderson Cancer Centre, Houston, TX 77030, U.S.A.
2
Present address: Pathology Department, Fundació Institut de Recerca Hospital Vall d’Hebrón, 08035 Barcelona, Spain
3
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
10
A. I. MacDonald and others
Dlg is defined as a tumour suppressor and is an essential septate
junction (similar to tight junctions) structural protein, which
also acts as a cell growth regulator and has an important role
in cell-membrane integrity, cell differentiation and cell polarity
[25]. hDlg is located at intercellular contact sites in epithelial
cells where it can bind to other cellular proteins including
APC (adenomatous polyposis coli), another tumour suppressor,
involved in the regulation of the Wnt signalling pathway [26,27].
The structure of hDlg is similar to that of ZO-1. It contains
several protein-interaction domains including an SH3 domain,
a HOOK domain, a GUK domain and three PDZ domains
[26]. However, it also contains an N-terminal protein-interaction
domain [28].
Cervical cancer progression is caused by persistent infection
with anogenital-infective ‘high risk’ HPVs [29]. The W12 model
of cervical cancer progression [12] was established from an
immortal but untransformed HPV16-positive cervical epithelial
cell line, W12 20861 (named W12G here) [30], and comprises two
derivative cell lines, W12GPX and W12GPXY [12]. We showed
previously that in the parental cell line, W12G, Cx43 localizes
to cell junctions, providing extensive GJIC, but in W12GPXY
cells that are fully transformed and invasive, Cx43 relocates from
the membrane to the cytoplasm, and these cells are deficient in
GJIC [12]. In the present study, we demonstrate hDlg relocation
to the cytoplasm in W12GPXY cells where it co-localizes with
Cx43. hDlg can co-immunoprecipitate Cx43 in cervical epithelial
cells and interact directly with Cx43 in vitro. Cx43 binds by its
C-terminal domain to both the N- and C-termini, but not the
PDZ domains of hDlg. siRNA (small interfering RNA) depletion
of hDlg showed that hDlg seems to maintain a cytoplasmic
pool of Cx43, possibly antagonizing lysosomal degradation of
this protein.
EXPERIMENTAL
Cell and culture conditions and drug treatment
W12G (clone 20861) cells are immortal but non-transformed
cervical epithelial cells [30]. They were grown using two different
culture methods, either grown on a feeder layer of mitomycin Ctreated 3T3 fibroblasts in F-medium [30] or without a fibroblast
feeder layer in KGM (keratinocyte growth medium) (Lonza).
Culturing of 3T3 cells was in DMEM (Dulbecco’s modified
Eagle’s medium) containing 10 % (v/v) DCS (donor calf serum)
(Invitrogen). W12G cells were seeded on to 3T3 monolayers at
2×105 per 10 cm dish and grown for up to 10 days with media
changes at 2 day intervals. 3T3 fibroblasts were removed by
trypsinization, and W12 cell layers were washed twice with PBS
before any further procedures were carried out. For immunofluorescence, W12G cells were grown in serum-free KGM until
colonies formed. W12GPXY cells, derived from W12G cells,
are fully transformed keratinocytes with invasive properties [12].
They do not require feeder cells or mitogens for growth and were
routinely cultured either in DMEM containing 10 % (v/v) FBS
(fetal bovine serum) or in KGM for immunofluorescence. However, in case the culture conditions altered the cellular phenotype,
experiments comparing W12G and W12GPXY cells were carried
out with cells grown in both F-medium and in KGM. CaSki,
cervical tumour cells, were also cultured in DMEM containing
10 % FBS. Cell lines were maintained at 37 ◦ C in a 5 % (v/v) CO2
humidified incubator. Lysosomal inhibition was achieved using
either 10 mM NH4 Cl or 200 μM chloroquine and incubation for
8 h at 37 ◦ C. Solution vehicle alone was added to mock-treated
cells. All chemicals were purchased from Sigma–Aldrich.
c The Authors Journal compilation c 2012 Biochemical Society
Co-IP (immunoprecipitation)
Cells at a confluency of 70 % were washed twice with ice-cold
PBS and scraped into 5 ml of chilled IP buffer [50 mM Tris/HCl,
pH 8.0, 0.5 % NP-40 (Nonidet P40), 150 mM NaCl, one tablet
of PhosSTOP phosphatase inhibitor cocktail (Roche) and one
tablet of CompleteTM mini protease inhibitor cocktail (Roche) per
10 ml]. Cell lysates were incubated on ice for 30 min and passed
through an 18-gauge syringe needle then cleared of cellular debris
by centrifugation at 12 000 g for 10 min at 4 ◦ C. Protein concentration was determined by Bradford’s assay (Bio-Rad Laboratories).
Cell extracts were pre-cleared with Protein G–Sepharose beads
(Sigma) for 1 h at 4 ◦ C. Primary antibodies {negative control
antibody, 1 μg of anti-SRPK1 polyclonal antibody clone 11305
(Santa Cruz Biotechnology); anti-hDlg polyclonal antibody H60
(Santa Cruz Biotechnology); and anti-ZO-1 polyclonal antibody
clone ZMD436 (Invitrogen)} were then added to 100 μg of
protein from each cell lysate and incubated for 2 h at 4 ◦ C with
agitation. Subsequently, fresh Protein G–Sepharose was added
and the volume was adjusted to 400 μl. The samples were mixed
by rotation at 4 ◦ C overnight. Immunocomplexes were harvested
by centrifugation (800 g, 4 ◦ C, 5 min) and washed five times
with 500 μl of ice-cold IP buffer. Proteins were solubilized by
adding protein-loading buffer [125 mM Tris/HCl, pH 6.8, 4 %
(w/v) SDS, 20 % (v/v) glycerol, 10 % (v/v) 2-mercaptoethanol
and 0.2 % Bromophenol Blue], boiled for 5 min and resolved by
SDS/PAGE followed by Western blotting.
Western blotting
Cells were scraped into protein-loading buffer containing
PhosSTOP phosphatase inhibitor cocktail and CompleteTM mini
protease inhibitor cocktail and passed through an 18-gauge
syringe needle. Proteins (10 μg) were resolved by SDS/PAGE
and subsequently transferred on to nitrocellulose membrane. For
standard Western blotting, membranes were blocked for 1 h at
room temperature (20 ◦ C) in 5 % (w/v) dried skimmed milk
powder dissolved in PBST (PBS containing 0.1 % Tween 20),
before overnight incubation at 4 ◦ C with primary antibodies
diluted in the same buffer. HRP (horseradish peroxidase)conjugated secondary antibodies [anti-(mouse IgG) or anti(rabbit IgG); GE Healthcare] were diluted 1:2000 in PBST
containing 5 % (w/v) skimmed milk powder and incubated for
1 h. Following washing in PBS, the blot was developed using
an ECL (enhanced chemiluminescence) kit (Thermo Scientific)
and exposed to Kodak X-OMAT film. For co-IP, a CleanBlot
IP detection kit (Thermo Scientific) was used. Western blotting
was carried out exactly as detailed in the manufacturer’s
protocol. Blots were blocked in StartingBlockTM blocking buffer
(Thermo Scientific) for 1 h before overnight incubation with
antibody diluted in StartingBlockTM buffer. The proprietary HRP
CleanBlot IP detection reagent that detects only the native
antibody was used. Anti-Cx43 rabbit polyclonal antibody was
purchased from Sigma–Aldrich (C-6219). A mouse monoclonal
antibody against ZO-1 was purchased from BD Transduction
Laboratories (610966). Mouse monoclonal clone 2D11 and rabbit
polyclonal clone H-60 anti-hDlg antibodies were purchased
from Santa Cruz Biotechnology. Anti-SRPK1 mouse monoclonal
antibody clone 12 was purchased from BD Biosciences and
goat polyclonal antibody clone sc11305 was from Santa Cruz
Biotechnology. Anti-E6 antibody was obtained from Arbor Vita
Corporation, Switzerland and anti-GAPDH (glyceraldehyde-3phosphate dehydrogenase) antibody was from AMS Biodesign
International. Anti-GST (glutathione transferase) antibody was
from Sigma. All of these antibodies were used at 1:1000
hDlg binds and regulates connexin 43
dilution. Anti-Cx26 and anti-Cx30 antibodies (Zymed) and antiE-cadherin antibody (BD Transduction Laboratories) were used
at 1:500. hDlg clone 2D11 was used at 1:250. HPV16 E2 TVG
261 polyclonal rabbit antibody was used at 1:5000. Quantification
of levels of proteins was carried out using Image J and normalized
to levels of GAPDH detected on the same membrane. A Student’s
t test was used to calculate statistical significance.
Plasmid construction
The human Cx43-containing plasmid pcDNA3-Cx43 (kindly
provided by Dr Dale Laird, Department of Anatomy and Cell
Biology, University of Western Ontario, London, ON, Canada)
was used as the template for PCR-mediated site-directed
mutagenesis. Primers were used to generate a C-terminal
portion of Cx43 corresponding to amino acids 263–382,
with or without the last five amino acids (PDZ-binding
motif) [17,18]. Both sense (5 -GGGAAAGGGGGATCCATGCAAAAATATGCTTAT-3 ) and antisense wild-type (5 -GGGAAAGGGGGATCCCTAGATCTCCAGGTCATCAGGCCG3 ) and antisense 5 (5 -GGGAAAGGGGGATCCCTAAGGCCGAGGTCTGCTGCTGGCACG-3 ) primers contained a
BamHI site (underlined) and a 9 nt flanking sequence. PCR
reactions were carried out using the Expand High Fidelity
PCR System (Roche) according to the manufacturer’s protocol.
BamHI-cut PCR fragments were ligated into calf-intestinal
phosphatase-treated, BamHI-cut pGEXT2T (GE Healthcare),
a GST expression vector. Ligated plasmids were transformed
into DH5α Escherichia coli and clones were analysed by
restriction digestion. Positive clones were confirmed by DNA
sequencing. FLAG–Cx43 was constructed by subcloning the
C-terminal portion of Cx43 into the bacterial expression vector
pT7-FLAG-1 (Sigma P1118). Specifically, a HindIII/BamHI
C-terminal Cx43 fragment was cut from a derivative of the vector
p3XFLAGCMV10 (Sigma) which contained the C-terminal
portion of Cx43 cloned between the HindIII and BamHI sites.
This fragment was then subcloned into the HindIII/BglII site of
pT7-FLAG-1. Ligated plasmids were transformed into DH5α E.
coli and clones were analysed by restriction digestion. Positive
clones were confirmed by DNA sequencing.
GST-protein expression and purification
Plasmids expressing GST-fusion proteins were transformed by
electroporation into E. coli BL21. Protein expression was under
the control of an IPTG (isopropyl β-D-thiogalactopyranoside)
inducible tac promoter. Cultures were grown to a D600 of 0.4–
0.6 before being induced for 3 h at 30 ◦ C with 1 mM IPTG. Cells
were pelleted by centrifugation at 5500 g at 4 ◦ C for 5 min. Cell
pellets were resuspended in 2 ml of PBS/1 % Triton X-100 and
suspensions were sonicated for 10×30 s on ice. Cellular debris
was removed by centrifugation at 12 000 g at 4 ◦ C for 15 min and
protease and phosphatase inhibitors were added to the supernatant.
Proteins were batch-purified using Glutathione Sepharose 4B (GE
Healthcare). Briefly, 500 μl of a 75 % slurry was added to each
sample and then incubated by rotation for 1 h at 4 ◦ C to allow GSTfusion-binding to the beads. Samples were washed with 5×10 ml
of wash buffer, then beads were pelleted by centrifugation (500 g,
4 ◦ C, 5 min). Beads were resuspended in 1 vol. glycerol and stored
at − 80 ◦ C. Purity of the proteins was assessed by SDS/PAGE.
FLAG protein expression and purification
FLAG–Cx43 constructs were transformed into BL21(DE3)-T1R
competent E. coli (Sigma). This strain expresses an IPTG-
11
inducible T7 polymerase and allows high-level induction and
expression of genes driven by the T7 promoter. Expression was
induced with 1 mM IPTG for 3 h at 30 ◦ C, as described above.
FLAG-tagged protein was captured on anti-FLAG M2 affinity
resin (Sigma) and purified by elution with FLAG peptide (Sigma).
His-tagged protein expression and purification
His–HPV16 E2 was expressed in BL21 E. coli and purified as
described using His·Bind resin (Novagen) [31].
GST-pull-down and in vitro binding experiments
GST–hDlg preparation and purification was carried out as
described previously [32]. GST–Cx43 C-terminal domain was
purified from bacterial lysates by a very similar protocol. Protein
purity and concentration was assessed using a Protein 200 LabChip kit (Agilent Technologies). Similar amounts of purified
protein (determined by Coomassie Blue visualization) were added
to 50–100 μg of protein extract in IP buffer and incubated for 2 h
with rotation at 4 ◦ C. Unbound protein was removed by repeated
washing in IP buffer and pellets were resuspended in proteinloading buffer. Samples were boiled at 100 ◦ C for 5 min before
being resolved by SDS/PAGE. For in vitro binding studies, equal
amounts (5 μg) of purified GST–hDlg and His–Cx43 were mixed
and rocked in 1 ml of IP buffer for 3 h at 4 ◦ C. Beads were washed
and samples eluted as above. Interacting proteins were detected
by Western blotting using specific antibodies.
Confocal immunofluorescence microscopy
Cell were grown on sterile 16 mm diameter coverslips until
70 % confluent, washed three times with PBS and fixed and
permeabilized with 100 % ice-cold methanol for 5 min at − 20 ◦ C
or with 2 % sucrose and 4 % formaldehyde for 10 min at room
temperature. Methanol fixation generally yielded better images
with the antibodies we used. The coverslips were blocked with
5 % (v/v) horse serum in PBS for 30 min at room temperature.
Cells were incubated with primary antibodies diluted 1:1000
(anti-Cx43; a gift from Dr Edgar Rivedal, Department of
Cancer Prevention, Institute for Cancer Research, Oslo University
Hospital, Oslo, Norway), 1:500 [anti-MPR (mannose 6-phosphate
receptor), Ab 2733, Abcam; anti-Cx26 and anti-Cx30, Zymed;
and anti-E-cadherin, Santa Cruz Biotechnology], 1:250 (ZO-1,
clone ZMD437; Invitrogen), 1:100 (β-catenin; BD Transduction
Laboratories) or 1:25 (hDlg, sc-9961; Santa Cruz Biotechnology)
in 5 % (v/v) horse serum in PBS for 1 h at room temperature.
Coverslips were washed in PBS six times before incubation for
1 h with secondary antibodies (diluted 1:200) conjugated to Alexa
Fluor® 488 or Alexa Fluor® 555 (Invitrogen). After washing in
PBS six times then once in water, coverslips were dried then
mounted with Vectashield hard-set mounting medium [with a
DAPI (4 ,6-diamidino-2-phenylindole) nuclear stain]. Negative
controls (no primary antibody) were included in all experiments.
Images were captured using either a Leica SP2-AOBS or Zeiss
LSM 510 confocal microscope. For the lysosomal inhibition
experiments, W12GPXY cells were either mock-treated or treated
with 10 mM NH4 Cl for 8 h and then incubated with Lysotracker
Red DND-99 (Invitrogen) in pre-warmed medium (100 nM) for
2 h. Cells were fixed (2 % sucrose and 4 % formaldehyde),
permeabilized (10 % sucrose and 0.5 % NP-40 solution), blocked
(5 % horse serum) and incubated for 1 h at room temperature with
the primary antibodies rabbit anti-(mouse Cx43) and anti-hDlg.
Coverslips were incubated for 1 h with the secondary antibodies
(diluted 1:500) donkey anti-(mouse Alexa Fluor® 647) and
c The Authors Journal compilation c 2012 Biochemical Society
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A. I. MacDonald and others
Figure 1 ZO-1, hDlg and Cx43 are present on the plasma membrane of W12G immortal cervical cells, but are found in the cytoplasm of W12GPXY transformed
cervical epithelial cells
Confocal immunofluorescence microscopy showing the location of Cx43 and hDlg in (A) W12G cells (immortal but not transformed cervical epithelial cells) compared with (B) W12GPXY cells
(transformed cervical epithelial cells). W12GPXY cells grew in small colonies. The image shows some cells in close contact and some isolated cells. White arrowheads indicate examples of
co-localization of Cx43 and hDlg. Green, Cx43; red, hDlg. (C) Confocal immunofluorescence microscopy showing the location of ZO-1 and Cx43 in W12G cells. (D) Confocal immunofluorescence
microscopy showing the location of Cx43 and ZO-1 in W12GPXY cells. White, Cx43; green, ZO-1. In all cases, cells were counterstained with DAPI (blue). Scale bar, 10 μm.
donkey anti-(rabbit Alexa Fluor® 488) (Invitrogen). Coverslips
were air-dried then mounted with Vectashield hard-set mounting
medium with DAPI (H-1500, Vector Laboratories). Images were
captured using a Zeiss LSM 510 confocal microscope with
appropriate settings, and colocalization was analysed with Zeiss
software.
Cellular Triton X-100 fractionation assay
Cells were washed with ice-cold PBS and extracted in 0.5 ml
of ice-cold PBS/1 % Triton X-100 containing PhosSTOP
phosphatase inhibitor cocktail, CompleteTM mini protease
inhibitor cocktail and 1 mM PMSF, then left for 1 h on ice with
intermittent vortex-mixing before centrifugation at 10 000 g for
5 min at 4 ◦ C. The supernatant containing Triton-soluble fractions
was removed and the Triton X-100-insoluble pellets resuspended
in 500 μl of protein-lysis buffer. Equal volumes of fractions were
then resolved by gel electrophoresis followed by immunoblotting.
Scrape-loading of cells
Cells were grown to confluence in 60 mm dishes. Cell layers were
washed twice in PBS, then 3 ml of 0.05 % Lucifer Yellow was
added and gentle ‘rolling’ cuts were made with a round-ended
scalpel blade. After 4 min at 37 ◦ C, cells were washed five times
in PBS, fixed in formaldehyde and examined under a fluorescence
microscope.
c The Authors Journal compilation c 2012 Biochemical Society
RESULTS
The PDZ domain protein hDlg relocates from the membrane to the
cytoplasm of cervical tumour cells in concert with Cx43
We showed previously that Cx43 is detected in both the cytoplasm
and in the membrane, in typical gap junction plaques, in nontransformed W12G cervical epithelial cells. In contrast, in the
fully transformed W12GPXY epithelial cell line derived from
W12G, gap junction plaques were not observed and Cx43
relocated to the cytoplasm, concomitant with a loss of GJIC [12].
Scaffolding proteins have been postulated to take part in Cx targeting to the membrane. Cx43 has been shown to bind the middle
of three adjacent PDZ domains in ZO-1 protein [17,18]; Cx45
also interacts with the PDZ domains of ZO-1 [21,22] and Cx32
interacts with the C-terminal SH3 domain of hDlg, which has also
been identified as a potential binding partner for Cx43 [33].
We used confocal microscopy to investigate whether hDlg
or ZO-1 scaffolding proteins had any relationship with Cx43
relocalization in cervical epithelial cells. hDlg was mainly
identified on the cell membrane and the cell margin in
untransformed W12G cells, partially co-localizing with Cx43
(Figure 1A). Cx43 was located in discrete regions of the plasma
membrane with a morphology indicative of gap junctions. In
addition, some cytoplasmic Cx43 and hDlg was observed.
In contrast, Cx43 was consistently located in the cytoplasm of
transformed W12GPXY cells and little cell membrane staining
was found (Figure 1B), as we observed previously [12]. Most
hDlg binds and regulates connexin 43
Figure 2
13
Triton X-100 cell extraction confirms Cx43 relocation in W12GPXY cells
(A) Western blot analysis of levels of various membrane-associated proteins in W12G and W12GPXY cells. The extracts used in the present study were isolated from the same batch of cells grown at
the same times as the extracts used in (B). Molecular mass in kDa is shown on the left-hand side. (B) Fractionation of Cx43, hDlg, ZO-1, GAPDH and E-cadherin into Triton X-100-soluble (S) and
-insoluble (I) fractions of W12G and W12GPXY cells. Cx43 is lost from the Triton X-100-insoluble fraction in W12GPXY cells compared with W12G cells. Several isoforms of hDlg can be observed
as expected [64]. (C) Quantification of relative total levels of hDlg, ZO-1, Cx43 and GAPDH protein in W12G cells (set at 100 %) compared with W12GPXY cells. Results are means +
− S.D. for three
independent experiments. **P < 0.005 (Student’s t test).
hDlg was also relocated to the cytoplasm of W12GPXY cells
and displayed a speckled pattern of staining. Moreover, in many
W12GPXY cells both hDlg and Cx43 were found in a perinuclear
location and some Cx43 and hDlg co-localized in the cytoplasm
(Figure 1B, white arrowheads). A similar pattern of Cx43 and
hDlg cytoplasmic location was observed in CaSki cells, another
cervical tumour cell line (results not shown). Next we examined
the location of ZO-1 in the two W12 cell lines as a positive control
for a PDZ domain protein known to bind Cx43. ZO-1 was more
difficult to detect than hDlg in W12G cells, but it displayed some
cell margin/membrane (white arrowhead) and some cytoplasmic
staining (Figure 1C). In the W12GPXY cells, ZO-1 gave a similar
pattern of staining. In contrast with the staining pattern observed
with hDlg, there was little evidence of perinuclear staining with
the anti-ZO-1 antibody (Figure 1D).
To confirm these data we carried out biochemical cell
fractionation using Triton X-100 extraction [34]. In this approach,
whole cells are transferred directly into Triton X-100 extraction
buffer, and soluble and insoluble fractions are obtained. Therefore,
because there is no pre-extraction input control, we used as ‘input’
whole-cell lysates in SDS/PAGE loading buffer from populations
of cells grown at the same time and under the same conditions as
those from which Triton X-100 extracts were made. Figure 2(A)
demonstrates input levels of the various proteins detected in the
Triton X-100 fractionation experiment in Figure 2(B). We found
signifcantly lower levels of Cx43 in W12GPXY cells than in
W12G cells. Figure 2(C) shows quantification of levels of Cx43
and the PDZ proteins hDlg and ZO-1 in both cell lines. In contrast
with Cx43, levels of hDlg and ZO-1 were similar and higher
respectively in the transformed cell line. Although ZO-1 was
difficult to detect by Western blotting (Figures 2A and 2B), it
was clearly present in W12G cells as shown by immunofluorescence in Figure 1(C). Figure 2(B) shows that in untransformed
W12G cells, Cx43 was extracted in both the soluble and
insoluble fractions, whereas in fully transformed W12GPXY
cells, Cx43 was detected only at a very low level in the insoluble
fraction and Cx43 levels were reduced overall (soluble plus
insoluble fractions) compared with W12G cells, reflecting the
40 % reduction in Cx43 level in W12GPXY cells compared with
W12G cells (Figure 2C). The Cx43 blot was subject to longer
exposure than the other blots in Figure 2(B) in order to visualize
any bands in the insoluble fraction from W12GPXY cells. hDlg
was found mainly in the Triton X-100-soluble fraction in both
W12G and W12GPXY cells. In contrast, ZO-1 was found in the
Triton X-100-insoluble fraction of both cell clones although levels
were much lower in W12G cells (Figure 2C). ZO-1 displayed
a cytoplasmic staining in W12GPXY cells that might indicate
removal from the plasma membrane so the retention of this
protein in insoluble fraction of W12GPXY cells indicates that it
must reside in cytoskeletal or membranous compartments of the
c The Authors Journal compilation c 2012 Biochemical Society
14
Figure 3
A. I. MacDonald and others
Cx relocation to the cytoplasm of W12GPXY cervical epithelial cells is specific for Cx43
W12G and W12GPXY cells were stained with antibodies against two other Cx proteins, Cx26 (A and B) and Cx30 (C and D). Confocal microscopy reveals membrane staining with each of these
antibodies in W12G and W12GPXY cells. Cells stained with anti-Cx antibodies (green) were co-stained with an anti-hDlg antibody (red). Cells were counterstained with DAPI (blue). White arrowheads
show membrane Cx26 and Cx30. Scale bar, 10 μm. Large merged images of a single cell in each multiple cell image (white arrows indicate the chosen cells) are shown on the right-hand side.
W12G cells can undergo Lucifer Yellow dye transfer following scrape loading (E), whereas W12GPXY cells do not transfer the dye efficiently (F), showing that W12GPXY cells do not possess good
gap junctional communication for molecules larger than 443 Da.
cytoplasm [35]. E-cadherin was extracted in both the soluble and
insoluble compartments [36], whereas GAPDH was also found
mainly in the soluble fractions as expected.
For both the biochemical fractionation and immunofluorescence experiments, each W12 cell line was grown both in
F-medium and in KGM. Very similar data were obtained
regardless of the growth conditions used for the W12 cell
clones. In case the relocation of Cx43 in W12GPXY cells was
due to a global down-regulation of gap junction formation, we
also examined localization of Cx26 and Cx30 in W12G and
W12GPXY cells. Figure 3(A) shows the distribution of Cx26 in
W12G cells. Not every cell expressed Cx26, but where Cx26 was
present it was found at the junctions between adjacent cells (white
arrowhead). Similarly, Cx30 was found on the membrane of some
c The Authors Journal compilation c 2012 Biochemical Society
W12G cells (Figure 3C, white arrowhead). For both of these Cxs
in the fully transformed W12GPXY cells, we observed increased
cytoplasmic and membrane staining with relatively large
membrane puncta compared with W12G cells (Figures 3B and
3D). Although the pattern of membrane staining with Cx26 and
Cx30 indicated possible formation of gap junction plaques, these
were not functional, at least as observed with Lucifer Yellow (443
Da) dye injection [12] and scrape loading. Figure 3(E) shows that
the dye was extensively spread into surrounding cells upon scrape
loading of W12G cells, but there was only limited dye transfer
when W12GPXY cells were used (Figure 3F).
In case there was some disruption of the plasma membrane,
perhaps due to the tumorigenic phenotype of W12GPXY cells
that could cause specific relocation of Cx43 we examined the
hDlg binds and regulates connexin 43
Figure 4
15
Cx43 relocation is not due to disruption of the W12GPXY cell membrane
Staining of W12G (A) and W12GPXY (B) with antibodies against two membrane proteins, E-cadherin and β-catenin. Cells stained with E-cadherin (red) were co-stained with an antibody against
Cx43 (green). β-Catenin is shown with green staining. Cells were counterstained with DAPI (blue). Scale bar, 10 μm.
location of E-cadherin and β-catenin (Figure 4). Both proteins
were observed at the cell periphery in both W12G and W12GPXY
cells although cell–cell contacts were not as smooth in
W12GPXY cells compared with W12G cells (see β-catenin
staining pattern).
Interaction of Cx43 with hDlg is detected in vivo in W12 cervical
epithelial cells
To investigate whether relocation of hDlg with Cx43 into the
cytoplasm might represent a direct protein–protein interaction
we first performed a series of co-IP experiments. Because
the molecular mass of Cx43 at 43 kDa is close to that of the
antibody heavy chain fragment we carried out the experiment
using the CleanBlot system (Thermo Scientific) that detects
only the antibody used in Western blotting and does not detect
heavy and light chain antibody fragments. Figure 5(A) shows
precipitation of Cx43 specifically with hDlg in W12G (lane 3)
and W12GPXY cells (lane 6). Anti-SRPK1 antibody (lanes 2 and
5) was used as an additional non-specific control to beads alone
(lanes 1 and 4). hDlg and SRPK1 were capable of precipitating
themselves in the reaction (Figures 5B and 5C). Figure 5(D) shows
GAPDH probing of the supernatants from the co-IP experiment
as an estimate of input. The results of a standard co-IP and
Western blotting protocol are shown in Supplementary Figure
S1 at http://www.BiochemJ.org/bj/446/bj4460009add.htm.
Cx43 cytoplasmic tail binds hDlg directly in vitro
Having shown that hDlg and Cx43 can exist as part of a complex
in epithelial cells, we next wanted to determine whether there
was any potential direct interaction between the two proteins.
We focused upon the C-terminal tail of Cx43, because this
domain is cytoplasmic and appears to mediate regulatory protein–
protein interactions [4]. To do this, we conducted in vitro
binding experiments between bacterially expressed and purified
proteins (Supplementary Figure S2 at http://www.BiochemJ.
org/bj/446/bj4460009add.htm). GST–hDlg was incubated with
a Cx43 C-terminal domain carrying an N-terminal FLAG-tag
(FLAG–Cx43CT) purified from uninduced or induced bacterial
lysates. As a control, we incubated FLAG–Cx43CT with an
irrelevant GST-tagged protein, GST–TopBP1 (topoisomerase
binding protein 1). Bound proteins were eluted and fractionated by
SDS/PAGE followed by Western blotting. GST–hDlg (Figures 5E
and 5F, lane 4), but not GST–TopBP1 (Figure 5E, lane 2), pulled
down the C-terminal domain of Cx43. With either GST–hDlg
or GST–TopBP1, no protein was precipitated using uninduced
bacterial FLAG lysate (Figure 5E, lanes 1 and 3). Similar amounts
of GST proteins were used in each reaction (Figure 5G). GST–
TopBP1 was capable of protein–protein interaction, because it was
able to precipitate the HPV16 E2 protein (Figure 5H), a known
TopBP1 interactor [37].
Cx43 cytoplasmic tail binds the N- and C-terminal domains, but not
the PDZ domains of hDlg
ZO-1 has many structural and functional similarities with hDlg
(both proteins are in the same MAGUK protein subgroup)
[17]. ZO-1 binds Cx43 through its second PDZ domain, so
hDlg might be expected to bind Cx43 in a similar manner.
However, another Cx, Cx32, has been shown to interact with
the C-terminal SH3/HOOK domain of hDlg [24]. To determine
which domain of hDlg bound Cx43, GST-fusion proteins of
the N-terminus (NT: amino acids 1–122), the N-terminus plus
one PDZ domain (NT + 1PDZ), the three PDZ domains alone
(3PDZ) and C-terminal (CT) domains (SH, HOOK and GUK
domains) were prepared (Supplementary Figure S2). GST-pulldown experiments in W12GPXY cells indicated that, although
full-length hDlg precipitated a large proportion of endogenous
Cx43 from W12GPXY cell extracts (Figure 6B, lane 3), the
isolated PDZ domains could only interact very inefficiently
(Figure 6B, lane 6). We detected weak binding to the C-terminal
domain (Figure 6B, lane 7). However, binding was clearly
observed with the hDlg N-terminal domain (Figure 6B, lanes
4 and 5). The upper bands in some of the tracks are non-specific
proteins that interact with GST and react with the Cx43 polyclonal
antibody. To control for binding to the hDlg-3PDZ region, we
tested interaction with a known PDZ binder, HPV16 E6, which is
expressed in W12GPXY cells. E6 was precipitated as efficiently
with full-length hDlg (Figure 6C, lane 3) as with the 3PDZ
domains of hDlg (Figure 6C, lane 6) but not with the N- or Ctermini as expected. To ensure that the C-terminal domain of hDlg
that we prepared was capable of interacting with another protein,
we tested binding of Cx32, previously reported to interact with
this domain [24]. Figure 6(D) shows that Cx32 was able to bind
full-length hDlg (Figure 6D, lane 3) and the hDlg C-terminal
domain (Figure 6D, lanes 4 and 6), but not the 3PDZ domain
region (Figure 6D, lane 5). The upper band in lane 3 that interacts
with anti-Cx32 antibody is a contaminating cross-reacting protein
from the bacterial lysate.
In the above experiments, the C-terminal domain of Cx43
was used to demonstrate interaction with hDlg. The extreme
C-terminal sequence DLEI can bind the second PDZ domain
c The Authors Journal compilation c 2012 Biochemical Society
16
Figure 5
A. I. MacDonald and others
Direct interation of Cx43 and hDlg in cervical epithelial cells
(A) Co-IP of endogenous Cx43 from W12G and W12GPXY cervical epithelial cells using
antibodies to proteins named above the blots. beads, beads without added antibody used in co-IP.
SRPK1, control for non-specific antibody binding: goat antibody against SRPK1 used in coIP. hDlg, co-IP with rabbit anti-hDlg antibody. A CleanBlot detection system (Thermo Scientific)
was used to avoid visualization of contaminating antibody fragments from these blots. (B) The
upper half of the blot in (A) was probed with an anti-hDlg antibody to show IP of hDlg with its own
antibody. (C) A very similar blot with the same protein samples was probed with an anti-SRPK1
mouse monoclonal antibody to reveal IP of SRPK1. (D) A one-tenth volume of supernatants
from the co-IP experiment was electrophoresed and Western blotted with an anti-GAPDH
antibody to give an estimate of protein loading. (E) GST-pull-down of purified bacterially
expressed N-terminal-FLAG-tagged C-terminus (CT) of Cx43. GST–hDlg, purified, bacterially expressed, GST–hDlg fusion protein (lanes 3 and 4) or GST–TopBP1 (lanes 1 and 2)
used in pull-down of FLAG-tagged C-terminal Cx43 and probed with an anti-FLAG antibody. (F)
A second Western blot of the same reaction products probed with anti-Cx43 antibody. (G) The
blot in (E) stripped and reprobed with anti-GST antibody. (H) GST pull-down of GST–hDlg or
GST–TopBP1 with His-tagged HPV16 E2 protein and probed with HPV16 E2 antibody. Molecular
mass in kDa is shown on the left-hand side. Ab, antibody; I, FLAG-tagged protein purification
from induced bacterial cell lysate; U, FLAG-tagged protein purification from uninduced bacterial
cell lysate.
of ZO-1 [17]. Therefore we tested whether this motif was
required for binding of Cx43 to hDlg in GST-pull-down assays.
Figure 6(E) shows that, in W12GPXY cells, wild-type C-terminal
Cx43 bound efficiently to full-length hDlg (Figure 6E, lane 3).
Binding between hDlg and Cx43 was still observed when the last
five amino acids of Cx43, containing the putative PDZ-binding
domain were deleted (Figure 6E, lane 4). In contrast, the same
deletion abrogated interaction of Cx43 with ZO-1 in the same
experiment (Figure 6F, lane 4).
c The Authors Journal compilation c 2012 Biochemical Society
Figure 6 GST-pull-down analysis of regions of interaction between hDlg
and Cx43
(A) Diagram of the modular structure of hDlg showing the N-terminal domain (hatched box),
three PDZ domains (light grey and numbered 1, 2 or 3) stretching from amino acids (aa) 220 to
545 and the C-terminal (C-term) SH, HOOK and GUK domains (striped, dark grey and stippled
boxes respectively) stretching from amino acid 560 to the end of the molecule at amino acid 911.
(B) GST-pull-down of Cx43 from W12GPXY cell extracts. Lane 1, input, one-fifth volume of lysate
used in GST-pull-downs. Lane 2, GST alone, negative control. Lane 3, GST–hDlg fusion protein.
Lane 4, GST–hDlg N-terminal domain (NT) (amino acids 1–122). Lane 5, GST–hDlg N-terminal
domain plus the first PDZ domain (NT + 1PDZ). Lane 6, GST–hDlg-3PDZ, amino acids 220–545
containing the three PDZ domains. Lane 7, GST–hDlg-CT, amino acids 560–911 covering the C-terminal domain of hDlg. The blot was probed with anti-Cx43 antibody. Cross-reactive
antibody bands were observed above the Cx43 band in lanes 1, 2, 6 and 7. (C) A very
similar Western blot of the same samples was probed with an anti-HPV16 E6 antibody.
(D) GST-pull-down of Cx32 from W12GPXY cell extracts. Lane 1, input, one-fifth volume
of lysate used in GST-pull-downs. Lane 2, GST alone, negative control. Lane 3, GST–hDlg
fusion protein purified from bacterial cell lysates. The lower band indicated with a white
asterisk is Cx32. Lanes 1–6 were from the same gel. The blot has been cut (white line) to
remove a second GST–hDlg fusion protein purified from bacterial cell lysates, but not used in
the experiments in lanes 4–6. Lane 4, GST–hDlg-CT, amino acids 560–911 covering the
C-terminal domain of hDlg. Lane 5, GST–hDlg-3PDZ, amino acids 220–545 containing
the three PDZ domains. Lane 6, GST–hDlg-CT + 3PDZ domains. Cross-reacting non-specific
bands of higher molecular mass than 32 kDa can be observed in lanes 1, 3, 4 and 6.
(E) GST-pull-down of hDlg from W12GPXY cells using GST–C-terminal (CT) Cx43 or
hDlg binds and regulates connexin 43
Figure 7
17
hDlg and Cx43 relocate to the endosomal/lysosomal compartments in W12GPXY cells
Confocal immunofluorescence microscopy of the location of Cx43 (green) and hDlg (magenta) in W12GPXY cells co-staining using Lysotracker Red to detect the lysosomal compartment (red). Cells
were either mock-treated (A) or treated (B) with 10 mM NH4 Cl for 8 h to inhibit lysosomal degradation. Cells were counterstained with DAPI. The rightmost panels in (A) and (B) show colocalization
of Cx43 and hDlg calculated using Zeiss confocal software. Scale bar, 10 μm. (C) Western blot analysis of hDlg, Cx43 and GAPDH controls in W12G and W12GPXY cells either mock-treated ( − )
or treated ( + ) with 10 mM NH4 Cl as above. Molecular mass in kDa is shown on the left-hand side. (D) Quantification of Cx43 levels upon NH4 Cl treatment. (E) Quantification of hDlg levels upon
NH4 Cl treatment. Results in (D and E) are means +
− S.D. for three independent experiments. *P < 0.05 (Student’s t test). Cntrl, control.
Cx43 and hDlg are located in the endosomal/lysosomal
compartment in W12GPXY cells
The distribution pattern of Cx43 and hDlg in W12GPXY
cells suggested a possible location in the ER (endoplasmic
reticulum)/Golgi where newly synthesized or aberrantly folded
or mutated Cxs can be located [38]. However, co-staining with
a Golgi marker (GM 130) revealed that most Cx43 and hDlg
was not in the Golgi (results not shown). Cxs can be removed
from the plasma membrane by endocytosis then finally degraded
by the endo-lysosomes [39]. The perinuclear location of Cx43
GST–C-terminal Cx43 with a deletion of the final five amino acids (Cx435). (F) GST-pull-down
of ZO-1 from W12GPXY cells using GST-C-terminal (CT) Cx43 or GST-C-terminal (CT) Cx43
with a deletion of the final five amino acids (Cx435). Lane 1, input, one-tenth volume of
lysate used in GST-pull-downs. Lane 2, GST-pull-down using GST alone as a negative control.
Lane 3, GST–CT-Cx43, N-terminal GST-tagged C-terminal domain of Cx43 used to pull down
ZO-1- and hDlg-containing complexes. Lane 4, GST–CT-Cx435, N-terminal GST-tagged
C-terminal domain of Cx43 with the last five amino acids deleted used to pull down ZO-1- and
hDlg-containing complexes. Molecular mass in kDa is shown on the left-hand side in (B–E).
and hDlg in the cytoplasm of W12GPXY cells [12] could
also correspond to endosomal/lysosomal compartments. To test
this hypothesis, W12GPXY cells were stained with the antiEEA (early endosome antigen) and anti-MPR antibodies that
detect early and late endosomes respectively. Cx43 and hDlg
showed partial co-localization with both endosomal markers, in
a perinuclear location (results not shown). To test whether Cx43
or hDlg co-localized in the endosomal/lysosomal compartment
for degradation, W12GPXY cells were mock-treated or treated
with the lysosomal inhibitor NH4 Cl at 10 mM for 8 h. Cells
were then stained with Cx43 (green) and hDlg (magenta)
and location was examined in relation to Lysotracker Red
(red). In mock-treated cells, Cx43 and hDlg located to the
cytoplasm with a proportion in a concentrated perinuclear location
where some of the proteins co-localized with the lysosomes
(Figure 7A, rightmost two panels, and Supplementary Figure S3
at http://www.BiochemJ.org/bj/446/bj4460009add.htm). Upon
NH4 Cl treatment, cells were no longer stained with Lysotracker
Red as expected and hDlg moved from a perinuclear location
to a more diffuse cytoplasmic location with some located to the
c The Authors Journal compilation c 2012 Biochemical Society
18
A. I. MacDonald and others
cell margin (Figure 7B and Supplementary Figure S3). Although
some Cx43 remained perinuclear in NH4 Cl-treated cells, more
Cx43 was located throughout the cytoplasm compared with
untreated cells. However there was no apparent trafficking to
the membrane. Some hDlg and Cx43 co-localized in a diffuse
pattern in the cytoplasm (Figure 7B, rightmost panel). To test
whether Cx43 or hDlg were targeted for lysosomal degradation,
protein extracts isolated from W12G and W12GPXY cells mocktreated or NH4 Cl-treated were fractionated by SDS/PAGE and
probed with antibodies against hDlg and Cx43 (Figure 7C).
Quantification of three separate experiments revealed that NH4 Cl
treatment had little effect on the levels of either protein in W12G
cells (Figures 7D and 7E). In contrast, levels of both hDlg and
Cx43 increased significantly (P < 0.05) in the presence of NH4 Cl
in W12GPXY cells. Experiments were repeated with chloroquine
(200 μM) as the lysosomal inhibitor with very similar results (not
shown).
To investigate a functional impact of hDlg on cytoplasmic
Cx43 levels, W12GPXY cells were treated with siRNA against
hDlg. If hDlg was necessary for delivery of Cx43 to the endosomal/lysosomal degradation pathway, loss of hDlg might result
in increased levels of Cx43. Effective knockdown of hDlg was
obtained in every experiment (Figure 8A and Supplementary Figure S4 at http://www.BiochemJ.org/bj/446/bj4460009add.htm).
Levels of the control proteins E-cadherin and GAPDH were
unaffected by a reduction in hDlg levels (Figure 8B). However,
no increase in Cx43 was detected. In fact, Cx43 was consistently
present at reduced levels of approximately 50 % of the control
(control siRNA) in W12GPXY cells treated with siRNA against
hDlg (Figures 8A and 8B). These results suggest that hDlg is
required to maintain a part of the cytoplasmic pool of Cx43 in the
cervical tumour cells. Next, to investigate a possible contribution
of hDlg in lysosomal targeting of Cx43, levels of Cx43 were
determined in cells where hDlg levels were reduced by siRNA
treatment and where lysosomal degradation was inhibited. If
hDlg were involved in antagonizing Cx43 lysosomal trafficking
then lysosomal inhibition might overcome the loss of Cx43
caused by hDlg knockdown. As already shown in Figure 7(D),
upon lysosomal inhibition with NH4 Cl (Figure 8C, lanes 4 – 6)
levels of Cx43 in W12GPXY cells were always higher compared
with mock-treated cells (Figure 8C, lanes 1 – 3) confirming
lysosomal targeting of Cx43 in W12GPXY. However, although
hDlg knockdown in the presence of NH4 Cl was still able to
reduce levels of Cx43 (compare lanes 5 and 6) quantification
of three separate experiments (Figure 8D) revealed that less
depletion of Cx43 (31 % reduction compared with NH4 Cl control,
P = 0.056) occurred than in mock-treated cells (51 % reduction
compared with mock control, P = 0.01). As controls, E-cadherin
and GAPDH levels were unaffected by hDlg knockdown and drug
treatment (Figure 8C). Finally, we investigated whether hDlg had
any role in membrane trafficking of Cx43. Confocal microscopy
revealed that siRNA-mediated reduction in hDlg levels did not
result in appearance of membrane Cx43 (Figure 8E). These results
indicate that hDlg can maintain part of the cytoplasmic pool of
Cx43, possibly in opposition to lysosomal degradation.
DISCUSSION
The disorganization of epithelial junctions can lead to defective
cell–cell adhesion, loss of cell polarity and unregulated cell
proliferation, and therefore may represent a crucial step in
tumorigenesis. Cx43 is a common unit of stratified epithelial
cell gap junctions and, in keratinocytes in culture, its loss
from the plasma membrane leads to loss of GJIC [12,40].
c The Authors Journal compilation c 2012 Biochemical Society
Our studies demonstrate a relocation of Cx43 from the plasma
membrane to the cytoplasm upon tumorigenic transformation of
W12GPXY cervical epithelial cells. The relocation of Cx43 did
not appear to be due to a significant loss of cell–cell contact,
because other proteins were maintained at the cell membrane.
One important factor to be considered in our experiments
was the phenotype of the two cervical epithelial cell lines.
W12G cells are immortal epithelial cells representative of low
grade dysplastic cervical epithelial cells. Because they are not
transformed they are still capable of some differentiation. The
growth conditions we used in our W12G experiments generated a
mixed population of cells both undifferentiated and differentiating
[41]. We used this cell population because we and others have
shown previously that Cx43 is most highly expressed in normal
and dysplastic differentiating epithelial cells [42,43]. On the
other hand, W12GPXY cells are transformed and unable to
differentiate. Lack of cell–cell communication and reduction in
Cx43 expression could reflect the undifferentiated status of these
cells, a component of their tumorigenic phenotype. However,
the W12-derived transformed cell line, W12GPX, the precursor to
W12GPXY, that has also lost differentiation capacity was found
to display good GJIC and membranous Cx43 [12]. Therefore we
propose that loss of GJIC in W12GPXY reflects a late event in
cervical tumour progression [12] and not a loss of Cx expression
due to an inability of the cells to differentiate.
Our results demonstrate that Cx43 relocation to the cytoplasm
of W12GPXY cells is specific, as other Cx proteins, Cx26 and
Cx30, remained on the membrane. Indeed, there appeared to
be increased Cx26 and Cx30 on the membrane of W12GPXY
cells (Figure 3). It is possible that the progressed malignant
transformation of W12GPXY cells results in increased expression
or stability of Cx26 and Cx30. Studies have revealed such an
increase in breast cancer [44], ovarian tumours [45], melanoma
[46] and pancreatic tumours [47]. W12GPXY cells displayed
punctate membrane staining of these two Cxs, yet do not display
competent GJIC, at least as assayed by transfer of Lucifer Yellow
[12], consistent with the lower size exclusion of Cx26/Cx30 in
comparision with Cx43 gap junction channels. Only some W12G
cells in the cell populations studied in Figure 3 expressed
membrane Cx26 and Cx30, whereas others did not stain with
antibodies against Cx26 and Cx30. This heterogeneity is probably
due to the presence of both differentiating epithelial cells (high
levels of Cx26/Cx30 expression) and undifferentiated cells
(low levels of Cx26/Cx30 expression) in the culture [42,43].
Cx43 binds to the second PDZ domain of the MAGUK
protein ZO-1 and it has been proposed that this scaffolding
interaction may organize gap junctions at the plasma membrane
by facilitating insertion of Cxs to the edge of gap junction plaques
aiding the connexon/gap junction transition and maintaining gap
junction stability [18,21,48,49]. ZO-1 may also be involved in
facilitating gap junction disassembly [50,51]. Our results confirm
that ZO-1 can interact with Cx43 in cervical epithelial cell
extracts. However, we detected little co-localization of ZO-1 with
Cx43 in confocal microscopy of W12GPXY cells, and ZO-1 was
extracted mainly in the Triton X-100-insoluble fraction, whereas
Cx43 was found mainly in the soluble fraction of these cells.
Therefore ZO-1 is unlikely to regulate Cx43 trafficking to the
cytoplasm in the cervical tumour cells.
hDlg, another MAGUK protein, was previously shown to bind
Cx32 [24] and was also identified as a potential binding partner for
the Cx43 C-terminal tail in a MS/MS (tandem MS) analysis screen
[52]. In contrast with ZO-1, hDlg is a good candidate for regulating Cx43 in cervical epithelial cells. We observed co-localization
of the two proteins in confocal images and neither protein
was found in the Triton X-100-insoluble fraction in W12GPXY
hDlg binds and regulates connexin 43
Figure 8
19
hDlg is required to maintain a cytoplasmic pool of Cx43
(A) Western blot analysis of levels of Cx43 in W12GPXY cells mock transfected (lane 1), transfected with a control scrambled hDlg siRNA (lane 2) or transfected with hDlg siRNA (lane 3). Levels of
E-cadherin and GAPDH are shown as control proteins not affected by hDlg siRNA knockdown. (B) Quantification of levels of Cx43 relative to levels of GAPDH in the hDlg knockdown experiments.
Results are mean +
− S.D.for three independent experiments. *P < 0.05 (Student’s t test). (C) Western blot analysis of levels of Cx43 with E-cadherin and GAPDH as controls, in W12GPXY cells
mock-transfected (lanes 1 and 4), transfected with a control scrambled hDlg siRNA (control siRNA; lanes 2 and 5) or transfected with hDlg siRNA (lanes 3 and 6). At 48 h after transfection, cells were
mock-treated (lanes 1–3) or treated with 10 mM NH4 Cl for 8 h (lanes 4–6) before harvesting for protein isolation. (D) Quantification of the results in (C) showing means +
− S.D. for three independent
experiments. *P < 0.05 (Student’s t test). (E) Confocal immunofluorescence microscopy of W12GPXY cells showing the location of Cx43 (green) upon treatment for 48 h with control siRNA or
treatment with siRNA against hDlg. Nuclei are stained with DAPI (blue). Scale bar, 10 μm.
cells. Moreover, we have direct and indirect evidence that hDlg
interacts with the Cx43 C-terminal domain. The MAGUK family
proteins (hDlg, ZO-1, ZO-2, PSD-95/SAP 90, p55 and others)
all display a similar modular structure composed of two to three
PDZ domains, an SH3 domain and GUK domain. Interestingly,
hDlg also contains an N-terminal domain, which is not found
in other MAGUKs [53,54]. This N-terminal domain controls
subcellular location of hDlg [55]. Our studies indicate that the
C-terminal domain of Cx43 forms a complex with hDlg within
cervical epithelial cells. Moreover, it binds hDlg directly and
protein–protein association is through the hDlg N- and C-terminal
domains. The relatively weak interaction of hDlg and Cx43 with
either terminus in GST pull-downs implies that full interaction
requires simultaneous binding to both domains. Therefore Cx43
binds hDlg in a different manner to that through which it binds
ZO-1: whereas the second PDZ domain of ZO-1 is required
for Cx43 binding, hDlg-Cx43 binding does not require the PDZ
domains. In support of a different mode of interaction between
Cx43 and hDlg is our observation that the last five amino
acids of Cx43 that comprise the PDZ-binding domain, including
those required for ZO-1 binding [18], were dispensable for hDlg
interaction.
c The Authors Journal compilation c 2012 Biochemical Society
20
A. I. MacDonald and others
In a scaffolding model, the different domains of hDlg
serve as docking modules for interactions with different
proteins/polypeptides [56]. hDlg could function in the transport
cycle of Cx43 by providing a docking platform for molecules
involved in assembling or disassembling gap junctions. This is
similar to what has been proposed for Cx43 interaction with
ZO-1 and raises the intriguing possibility that both PDZ proteins
may co-operate to regulate gap junction trafficking.
The transport cycle of Cxs involves trafficking from the
ER/Golgi to the plasma membrane and formation of gap junction
plaques. Internalization of membrane Cxs may be mediated
through the ‘connexosome’ and targeted to either the lysosomal
or proteasomal degradation pathways. It has been proposed that
different populations of Cxs may be differentially targeted [4]. Our
results demonstrate a significant increase in lysosomal degradation of Cx43 in transformed W12GPXY cells compared with the
untransformed W12G cells. Although co-localization of hDlg and
Cx43 in W12GPXY cells suggested that hDlg might target Cx43
to the lysosomes, and indeed the two proteins do co-localize
with a lysosomal marker, siRNA knockdown of hDlg revealed
a decrease, rather than an increase in Cx43 levels. Our results
suggest that although hDlg may accompany a pool of Cx43 to
the lysosomes, some hDlg is required to maintain another pool of
Cx43 in the cytoplasm in transformed cervical epithelial cells. The
two pools of hDlg may represent different splice isoforms that can
be detected with the anti-hDlg antibodies we used [55,57] or differentially post-translationally modified forms of the protein [58].
There is conflicting evidence for the roles of Cxs in cancer. In
some cases, particularly during tumorigenesis, it is clear that Cxs
may act as tumour suppressors. However, at later disease stages,
Cxs may play a role in the various processes between cancer-cell
migration and metastasis [59]. W12GPXY cells have invasive
properties [12], and it is possible that an hDlg-maintained Cx43
pool in cervical tumour cells could serve to reform gap junctions
in cells disseminated to distant metastatic sites.
In conclusion, our findings highlight hDlg as a physiological
regulator of Cx43 and suggest that hDlg may contribute to control
of the Cx43 life cycle. It has been recognized for some time that,
in line with disruption of GJIC, hDlg is a common target in
epithelial cancers, displaying mutations, changes in protein levels
and changes in subcellular localization [60–62]. Although W12
cells express HPV16 E6 protein, which can bind and target PDZ
proteins for proteasomal degradation, it has only a minimal role in
hDlg degradation [63], meaning that E6 regulation of hDlg levels
is unlikely to control Cx43 relocation. We showed previously
a significant alteration in Cx43 isoform profile in W12GPXY
cells compared with W12G cells [12]. Dephosphorylation and
phosphoprotein purification studies have revealed that at least
some of these isoforms are due to altered phosphorylation (results
not shown). Since many sites of phosphorylation are located in
the C-terminal domain of Cx43 [8], they could modulate Cx43
interaction with hDlg or Cx43 trafficking leading to loss of GJIC
during malignant progression of keratinocytes. Future studies will
shed light on the relationship of the phosphorylation status of
Cx43 and alteration of its functions in these tumour cells.
AUTHOR CONTRIBUTION
Alasdair MacDonald carried out all of the experiments in Figures 2, 6 and 8, some of the
experiments in Figures 3, 4 and 7, and helped to write the paper. Peng Sun carried out
the experiments in Figures 1, 3 and 4. Hegel Hernandez-Lopez carried out the experiment in
Figure 7(A). Trond Aasen developed the W12GPXY cell line and carried out the experiment
in Figures 3(E) and 3(F). Malcolm Hodgins was a co-supervisor of Peng Sun and Trond
Aasen, a co-applicant on the grant that supported Alasdair MacDonald, gave advice on
experiments, and helped to write and revise the paper. Michael Edward was a co-supervisor
c The Authors Journal compilation c 2012 Biochemical Society
of Peng Sun and Trond Aasen. Sally Roberts had the original idea of Cx43–hDlg interaction
and gave advice on developing the project. Paola Massimi generated important background
data. Miranda Thomas generated most of the Dlg/E6 constructs. Lawrence Banks was a
co-applicant on the grant that supported Alasdair MacDonald, supervised Paola Massimi
and Miranda Thomas, gave advice on experiments, and helped to write and revise the
paper. Sheila Graham carried out the experiments in Figure 5, co-ordinated the project,
was a co-supervisor of Peng Sun and Trond Aasen, was the principal investigator on the
grant that supported Alasdair MacDonald, supervised his research, and wrote and revised
the paper.
ACKNOWLEDGEMENTS
We are grateful to Margaret Stanley (Department of Pathology, University of Cambridge,
Cambridge, U.K.) for the original W12 cell line and Paul Lambert (School of Medicine and
Public Health, University of Wisconsin, Madison, WI, U.S.A.) for the W12E and W12G
clones of this line. We thank Patricia Martin (School of Health and Life Sciences, Glasgow
Caledonian University, Glasgow, Scotland, U.K.) for generously sharing reagents. Mary
Donaldsdon (Centre for Virus Research, University of Glasgow, Glasgow, Scotland, U.K.)
kindly provided purified GST–TopBP1. We are very grateful to Colin Loney for technical
assistance with confocal microscopy. We thank Arbor Vita Corporation for provision of
the E6 antibody.
FUNDING
This work was funded by the Association for International Cancer Research [grant
number 08–0159 (to S.V.G., M.B.H. and L.B.)]. P.S. and T.A. were supported by Glasgow
University postgraduate scholarships and Overseas Research Studentship awards. H.H.-L.
is supported by a CONACyT (Consejo Nacional de Ciencia y Tecnologı́a) scholarship from
the Mexican Government.
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Received 15 July 2011/28 May 2012; accepted 1 June 2012
Published as BJ Immediate Publication 1 June 2012, doi:10.1042/BJ20111144
c The Authors Journal compilation c 2012 Biochemical Society
Biochem. J. (2012) 446, 9–21 (Printed in Great Britain)
doi:10.1042/BJ20111144
SUPPLEMENTARY ONLINE DATA
A functional interaction between the MAGUK protein hDlg and the gap
junction protein connexin 43 in cervical tumour cells
Alasdair I. MACDONALD*, Peng SUN*1 , Hegel HERNANDEZ-LOPEZ*, Trond AASEN*2 , Malcolm B. HODGINS†,
Michael EDWARD†, Sally ROBERTS§, Paola MASSIMI‡, Miranda THOMAS‡, Lawrence BANKS‡ and Sheila V. GRAHAM*3
*MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow,
Glasgow G12 8TT, Scotland, U.K., †Section of Dermatology, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G11 6NU, Scotland,
U.K., ‡International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy, and §CRUK Birmingham Cancer Centre, School of Cancer Sciences,
University of Birmingham, Birmingham B15 2TT, U.K.
Figure S1 Co-immunoprecipitation of hDlg and Cx43 using a standard
Western blotting protocol
Co-IP of endogenous Cx43 from W12G and W12GPXY cervical epithelial cells using antibodies
to proteins named above the blots. ZO-1 interaction with Cx43 was used as a positive control. The
blot was probed with rabbit anti-Cx43 antibody. Molecular mass in kDa is shown on the left-hand
side. The broad Cx43 band (indicated with a bar to the right-hand side of the blot) indicates
several Cx43 isoforms [1]. The antibody (Ab) heavy and light chain bands are indicated. The
multiple dark bands above the antibody heavy chain band are antibody-related fragments that
cross-react with the Cx43 antibody. Input, one-tenth volume of W12G supernatant used in the
co-IP. beads alone, beads without added antibody used in co-IP. SRPK1, control antibody against
SRPK1 used in co-IP. hDlg, co-IP with anti-hDlg antibody. ZO-1, co-IP with ZO-1 antibody.
The anti-hDlg and anti-ZO-1 antibodies used in co-IP were both rabbit antibodies, as was the
anti-Cx43 antibody used in the immunoblot, so antibody heavy and light chain fragments can
be observed at approximately 50 and 25 kDa respectively in lanes 4, 5, 8 and 9. The multiple
bands above 50 kDa are background bands. The SRPK1 negative control antibody was a goat
antibody so only the light chain antibody fragment can be observed in lanes 3 and 7. Lanes 4
and 8 demonstrate co-IP of endogenous Cx43 with hDlg from W12G and W12GPXY cell extracts
respectively. The Cx43 bands in these lanes migrate more slowly than that in the input lane,
probably as a consequence of the amount of antibody fragments present in the protein mixture
applied to the gel. Cx43 co-immunoprecipitated with ZO-1 in both cell lines (lanes 5 and 9).
No Cx43 co-IP was observed with Protein G beads alone or with SRPK1, the negative control
antibody.
1
2
3
Present address: MD Anderson Cancer Centre, Houston, TX 77030, U.S.A.
Present address: Pathology Department, Fundació Institut de Recerca Hospital Vall d’Hebrón, 08035 Barcelona, Spain
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
A. I. MacDonald and others
Figure S2
Purification of GST, FLAG and His-tagged proteins using in GST-pull-down experiments
(A) GST–hDlg full-length and truncated/mutated proteins. Red arrows indicate the full-length protein in each case. (B) Presence of N-terminal-FLAG-tagged C-terminal Cx43 protein in induced (I)
bacterial cells lysates, but not in uninduced (U) cell lysates. (C) Eluted (E) His-tagged HPV16 E2 protein. W, the last wash fraction prior to elution. (D) Purified GST–TopBP1 C-terminus protein and
GST–Cx43 C-terminal domain. Molecular mass in kDa is shown on the left-hand side in (A–C) and on the right-hand side in (D).
Figure S3
Co-localization of Cx43 and hDlg with the lysosomal compartment in W12GPXY cells
Images of cell populations triple-stained with antibodies against Cx43 (green) and hDlg (magenta) and Lysotracker Red (red) to stain the lysosomes in the presence and absence of 10 mM NH4 Cl.
Cells are counterstained with DAPI (blue). Scale bar, 10 μM.
c The Authors Journal compilation c 2012 Biochemical Society
hDlg binds and regulates connexin 43
Figure S4 Quantification of levels of hDlg knockdown in the experiments
in Figure 8 in the main text
Results are mean +
− S.D. for five independent experiments. Cntrl, control.
REFERENCE
1 Aasen, T., Hodgins, M. B., Edward, M. and Graham, S. V. (2003) The relationship between
connexins, gap junctions, tissue architecture and tumour invasion, as studied in a novel in
vitro model of HPV-16-associated cervical cancer progression. Oncogene 22, 7969–7980
Received 15 July 2011/28 May 2012; accepted 1 June 2012
Published as BJ Immediate Publication 1 June 2012, doi:10.1042/BJ20111144
c The Authors Journal compilation c 2012 Biochemical Society